This application is a National Stage Application under 35 U.S.C. xc2xa7371 of PCT/FR99/01207 filed May 21, 1999. This application claims priority under 35 U.S.C. xc2xa7119 to France 98/06540 filed May 25, 1998, the entire contents of which are herein incorporated by reference.
The present invention relates to molecular rods, to their uses in a method of attaching and/or crystallizing macromolecules, to the resulting products and to the applications of said products in the field of materials and structural biology, especially as biosensors or biomaterials.
Knowledge of the structure of proteins and especially their active sites is essential to an understanding of their mechanism of action. Several methods are available for conducting such studies: X-rays, NMR and electrocrystallography (2D crystallization).
To perform the actual crystallization, the technique of two-dimensional crystallization on a lipid monolayer or film at the air/water interface (E. E. Ugziris et al., Nature, 1983, 301, 125-129) makes it possible to form auto-organized systems of biological macromolecules (crystals) and determine the structures of these molecules by electron microscopic analysis of the crystals obtained.
This method consists in creating a lipid monolayer at an air/liquid interface, the lipids being selected to interact with the proteins, present in the liquid phase, which attach themselves to the lipids and then form an organized network.
Attachment of the proteins to the lipids of the monolayer involves chemical interactions at the polar head of the lipids. These interactions are either nonspecificxe2x80x94in which case the lipids possess charged polar ends giving rise to crystallization by ionic interactionsxe2x80x94or specific. In the latter case, the polar head of the lipids carries ligands with a strong affinity for the proteins to be attached.
In particular, it has been possible to show that soluble proteins can crystallize two-dimensionally on charged lipid films or lipid films functionalized by a ligand of the protein studied (B. J. Jap et al., Ultramicroscopy, 1992, 46, 45-84).
More recently, lipids functionalized by metal complexes such as nickel complexes (E. W. Kubalek et al., J. Struct. Biol., 1994, 113, 117-123) have made it possible to crystallize so-called histidine-tagged fusion proteins. These proteins in fact possess a sequence composed of several histidines at their N-terminal or C-terminal end. It has been possible to show that the attachment of such proteins to a nickel-functionalized lipid is due to a strong interaction between the nickel complex and the polyhistidine sequence (C. Vxc3xa9nien-Brian et al., J. Mol. Biol., 1997, 274, 687-692). Such functionalized lipids have enabled crystallization to be achieved, especially in cases where the appropriate ligand was not available.
However, the crystallization of proteins on lipid films has the disadvantage of being relatively random and of being dependent on numerous factors which cannot easily be controlled simultaneously:
The ligand carried by the lipids must be sufficiently accessible to be able to interact with the proteins. This accessibility depends on the length of the spacer arm between the lipid and the ligand: if it is too short, it allows the protein to penetrate inside the lipid layer; if it is too long, it gives the bound protein too great a degree of freedom and increases the incidence of defects in the crystal.
The lipid monolayer must be sufficiently fluid to give the bound protein a sufficient lateral and rotational mobility, thereby enabling the proteins to organize themselves relative to one another and develop intermolecular contacts so as to produce the crystal.
Another difficulty inherent in crystallization on a lipid monolayer concerns the stability of the monolayer; in fact, the stability of the air/liquid interface is difficult to control. In addition, the lipid monolayer must remain stable, not only before but also after attachment of the proteins, in order to allow spatial organization of the proteins.
For the microscopic study, which follows the crystallization step, it is necessary to create a large number of planes because of the planar nature of the structure obtained.
Consequently, the Inventors set out to provide structures, hereafter called molecular rods, which were suitable for attaching and crystallizing biological macromolecules in solution, as well as a method of attaching said biological macromolecules in solution and optionally of inducing their auto-organization, said method satisfying the practical needs better than the 2D crystallization methods used in the prior art.
The present invention relates to molecular rods, characterized in that they have a structure represented by the following general formula I: 
in which:
P is a polymer selected from the group comprising polyphenylenes, polyphenylenevinylenes, polyphenyleneethynylenes and polyvinylenes, as illustrated by the formulae below: 
in which:
A is a hydrogen atom or one of the following groups: alkyl, OH, O-alkyl, NH2, NH-alkyl, CO2H, CO2-alkyl, CONH2, CONH-alkyl;
GpF (functional group) is a group Bxe2x80x94R, in which:
B (bonding arm) is selected from C1-C10 hydrocarbon groupings which are optionally substituted by alkyl groups, may or may not have units of unsaturation or polyoxyethylene units and may or may not have phosphate groups in the middle of the chain, such as: 
in which:
m is an integer from 1 to 10, and
X is O, NHCO, OCO, COO, CONH, S, CH2 or NH and constitutes, at the ends of said hydrocarbon groupings, organic coupling groups of the ester, amide, ether or thioether type; and
R is a hydrophilic group selected from positively or negatively charged groups; ligands or analogues of biological macromolecules such as, without implying a limitation, biotin, novobiocin, retinoic acid, steroids or antigens; or organometallic complexes interacting with amino acids or nucleic acids, such as complexes of copper, zinc, nickel, cobalt, chromium, platinum, palladium, iron, ruthenium or osmium with ligands like IDA, NTA, EDTA, bipyridine or terpyridine, said ligands optionally being functionalized by alkyl groups for bonding to E (at X); without implying a limitation, positively or negatively charged groups are understood as meaning ammonium, carboxylate, phosphate or sulfonate groups; the following groups may be mentioned as examples: xe2x80x94N(CH3)3+ or xe2x80x94CO2xe2x88x92;
n is an integer between 5 and 1000;
p is an integer between 0 and 10; and
E (spacer segment) is a chemical unit whose nature does not disturb the rigid structure of the skeleton formed by P, and is a phenylene, ethynylene or vinylene unit or a combination of these units, such as phenyleneethynylene, as illustrated by the formula below: 
in which A is a hydrogen atom or one of the following groups: alkyl, OH, O-alkyl, NH2, NH-alkyl, CO2H, CO2-alkyl, CONH2, CONH-alkyl.
Together with GpF and E, the different polymers P as defined above give the following formulae: 
In terms of the present invention, alkyl is understood as meaning linear or branched or optionally substituted C1-C6 alkyl groups.
The substituents of the C1-C10 hydrocarbon groupings B are selected particularly from C1-C6 alkyls.
Polymers whose skeleton has a large number of units of conjugation (polyphenylene, polyphenylenevinylene, polyphenyleneethynylene) have already been described (Angew. Chem. Int. Ed., 1998, vol. 37, pp. 402-428) and are used for their electronic and fluorescent properties in non-linear optics (Macromolecules, 1994, 27, 562-571 and J. Phys. Chem., 1995, 99, 4886-4893).
The polymers according to the present invention are functionalized by groups GpF which, in association with the moiety E, give the molecular rod according to the invention particular properties:
it is linear, rigid and soluble in aqueous media;
it is regularly functionalized by groups with a very strong affinity for biological macromolecules; and
when dissolved with a biological macromolecule, it is particularly suitable for the attachment and/or auto-organization of said macromolecules to/on said rod by molecular recognition.
The structure of the molecular rods according to the invention is illustrated in FIG. 1:
P constitutes a polymer skeleton, which must be rigid and linear overall so as to have the character of a molecular rod; and
E makes it possible to control the distance L2 between the functional groups GpF, while the bonding arm B of GpF makes it possible to control the distance L1 between the group R and the axis of the polymer, as illustrated in FIG. 2.
In one advantageous embodiment of said molecular rods, they have the following general formula II: 
in which:
p=0: absence of E;
P is the group b as defined above;
GpF comprises a group B represented by a group ƒ as defined above in which m=3, one of the groups X is NHCO and the other is CH2, and a group R represented by a nickel-based organometallic complex (Nixe2x80x94NTA complex); and
n is an integer between 5 and 1000.
In another advantageous embodiment of said molecular rods, they have the following general formula III: 
in which:
m is an integer between 1 and 10;
p is an integer between 0 and 10;
P is the group b as defined above;
GpF comprises a group B represented by a group h as defined above in which the two groups X are identical and are NHCO, associated with a group R represented by a nickel-based organometallic complex (Nixe2x80x94NTA complex) in which the ligand NTA is functionalized by a C4 alkyl group=(CH2)4; and
n is an integer between 5 and 1000.
The present invention further relates to a method for the attachment and/or auto-organization of biological macromolecules, characterized in that it comprises essentially the incubation of a biological macromolecule in solution with a molecular rod, as defined above, for at least 15 minutes under suitable temperature and pH conditions.
After attachment and/or auto-organization of the macromolecules, a supramolecular object is obtained.
In the auto-organization process according to the invention, the supramolecular object obtained will optionally be able to develop into a spiral crystal of biological macromolecules around the molecular rod.
Said method is particularly suitable for controlling the spiral crystallization of the biological macromolecules around said molecular rods.
Surprisingly, the molecular rods according to the invention which make it possible to attach biological macromolecules in solution and optionally to induce their auto-organization offer important applications in the fields of nanomaterials or structural biology:
attachment of biological macromolecules to the molecular rods, with or without control of the orientation of this attachment;
control of the spiral crystallization of the biological macromolecules around the molecular rods; and
structural study of the biological macromolecules by electron microscopic analysis of the spiral crystals obtained.
In one advantageous mode of carrying out said method, said biological macromolecules are especially soluble membrane or trans-membrane proteins, enzymes, antibodies, antibody fragments or nucleic acids.
In another mode of carrying out said method, said solution consists of an aqueous or aqueous-alcoholic solvent for solubilizing said biological macromolecules, optionally containing at least one detergent, depending on the biological macromolecule to be crystallized.
In another advantageous mode of carrying out said method, the incubation conditions are preferably as follows: incubation at room temperature for 15 minutes to 48 hours at a pH of between 5.5 and 8.5.
The method according to the present invention is particularly applicable to determination of the three-dimensional structure of soluble proteins.
The formation of a spiral crystal of a biological macromolecule, such as a protein, on a molecular rod is the result of a perfect match between the dimensions of the macromolecule (diameter) and the parameters of the rod (distances L1 and L2 and length of the molecular rod). The distance L1 represents the distance between the group R and the axis of the polymer. The distance L2 represents the distance between two groups R. The length of the molecular rod is equivalent to the degree of polymerization of the polymer (cf. FIG. 2).
Surprisingly, said method makes it possible to obtain arrangements of biological macromolecules which allow structural studies by electron microscopy or the preparation of novel nanomaterials useful for their physical, electrical or biological properties.
The present invention consequently includes the preparation of a library of molecular rods in which the distances L1 and L2 are variable.
The present invention further relates to a supramolecular object, characterized in that it consists of a molecular rod, as defined above, to which biological macromolecules are attached in a non-covalent manner or on which they are organized in a crystalline form.
The present invention further relates to the applications of said supramolecular object, and to the structural study of the macromolecules associated therewith, as a biological reagent and especially as an immunological reagent and as a biosensor or bioconductor.